The role of cellular antioxidant pathways in protecting neurons during photodynamic therapy

The effect of photodynamic therapy (PDT) on neurons is an important consideration when treating cancers within or adjacent to the nervous system. The photosensitiser, meta-tetrahydroxyphenyl chlorin (mTHPC) is effective in destroying tumour cells but can spare neurons in culture. The aim of this study was to investigate the role of cellular antioxidant pathways in protecting neurons from damage during mTHPC-mediated PDT. Exposing cells to 4 µg/ml mTHPC-mediated PDT in a 3D culture system resulted in substantial cell death among tumour cells and glia, but survival of neurons. To investigate this phenomenon, prior to PDT neurons were treated with drugs to block antioxidant pathways; L-buthionine sulfoximine (L-BSO) depleted glutathione, and diethyldithiocarbamate (DDC) and 2-methoxyoestradiol (2MeOE2) were used to inhibit superoxide dismutase SOD 1 and SOD 2 respectively. Neuronal cell death was assessed 24 h later. Neuronal death following mTHPC-mediated PDT was significantly increased in the presence of either DDC or L-BSO compared to PDT-only controls. There was a slight increase in neuronal death following PDT in neurons treated with 2-MeOE2, but this was not statistically significant. Overall, inhibition of glutathione resulted in 63 ± 7 % of neurons dying and inhibition of SOD 1 resulted in 42 ± 7 % neuron death. These results suggest that the mechanism by which neurons show reduced sensitivity to mTHPC-mediated PDT involves endogenous antioxidant pathways, the blocking of which results in neuronal cell death. Of the antioxidant pathways investigated here it is apparent that glutathione and SOD 1 play an important role, whereas inhibition of SOD 2 had little effect. Further studies are required to understand fully the cellular mechanisms involved in protecting neurons during this treatment, and the culture system described here is a useful tool for such research. Ultimately, understanding the conditions under which neurons may be able to survive PDT or other oxidative damage is important in developing strategies for treating tumours within or adjacent to the nervous system, where avoidance of neuronal damage has the potential to reduce side effects

Spectroscopic evaluation of QDs encapsulated with a novel biocompatible polymer for cancer diagnosis

Quantum dots (QDs) are new class of fluorescent inorganic nanocrystals which have been used for in vitro and in vivo imaging. Their unique optical properties such as broad excitation spectra, narrow emission spectrum and resistance to photobleaching make them ideal for biological labeling. Sentinel lymph node biopsy is a means of ultra-staging cancer metastasis and is now the standard of care in breast cancer surgery. Localisation of sentinel nodes is also important in the treatment of head and neck cancer. Current tracers for SLN biopsy include the blue dye have various limitations that could be overcome by quantum dots that emit in near infrared range (>700 nm). To safely deliver QDs they must be encapsulated in a biocompatible coating. In this study we encapsulate CdTe QDs with new nanocomposite material based on a silsesquioxane modified poly (carbonate-urea) urethane polymer, and evaluated their spectroscopic properties

Organic Nanodelivery Systems as a New Platform in the Management of Breast Cancer: A Comprehensive Review from Preclinical to Clinical Studies

Breast cancer accounts for approximately 25% of cancer cases and 16.5% of cancer deaths in women, and the World Health Organization predicts that the number of new cases will increase by almost 70% over the next two decades, mainly due to an ageing population. Effective diagnostic and treatment strategies are, therefore, urgently required for improving cure rates among patients since current therapeutic modalities have many limitations and side effects. Nanomedicine is evolving as a promising approach for cancer management, including breast cancer, and various types of organic and inorganic nanomaterials have been investigated for their role in breast cancer diagnosis and treatment. Following an overview on breast cancer characteristics and pathogenesis and challenges of the current treatment strategies, the therapeutic potential of biocompatible organic-based nanoparticles such as liposomes and polymeric micelles that have been tested in breast cancer models are reviewed. The efficacies of different drug delivery and targeting strategies are documented, ranging from synthetic to cell-derived nanoformulations together with a summary of the interaction of nanoparticles with externally applied energy such as radiotherapy. The clinical translation of nanoformulations for breast cancer treatment is summarized including those undergoing clinical trials

Liquid Crystalline Hydroxyapatite Nanorods Orchestrate Hierarchical Bone‐Like Mineralization

Bone matrix exhibits exceptional mechanical properties due to its unique nanocomposite structure of type I collagen fibrils and hydroxyapatite (HAp) nanoparticles in hierarchical liquid crystalline (LC) order. However, the regeneration mechanism of this LC structure is elusive. This study investigates the role of the LC structure of HAp nanorods in guiding aligned mineralization and its underlying molecular mechanism. A unidirectionally oriented LC phase of HAp nanorods is developed through engineering‐assisted self‐assembling. This is used to study the growth direction of long‐range aligned extracellular matrix (ECM) and calcium deposit formation during the osteogenic differentiation of human bone marrow‐derived mesenchymal stem cells. It is found that 2 key regulatory genes, COL1A1 and COL4A6, lead to the formation of aligned ECM. Activation of the PI3K‐Akt pathway enhances osteogenesis and promotes ordered calcium deposits. This study provides evidence for elucidating the mechanism of LC‐induced ordered calcium deposition at hierarchical levels spanning from the molecular to macro‐scale, as well as the switch from ordered to disordered mineralization. These findings illuminate bone regeneration, contribute to the development of biomimetic artificial bone with long‐range ordered structures, and suggest a basis for therapeutic targeting of microstructure‐affected bone disorders and the broader field of cell‐ECM interactions

Highly sensitive luminescence detection of photosensitized singlet oxygen within photonic crystal fibre

Highly sensitive, quantitative detection of singlet oxygen (1O2) is required for the evaluation of newly developed photosensitizers and the elucidation of the mechanisms of many processes in which singlet oxygen is known, or believed, to be involved. The direct detection of 1O2, via its intrinsic phosphorescence at 1270 nm, is challenging because of the extremely low intensity of this emission, coupled with the low quantum efficiency of currently available photodetectors at this wavelength. We introduce hollowcore photonic crystal fibre (HC-PCF) as a novel optofluidic modality for photosensitization and detection of 1O2. We report the use of this approach to achieve highly sensitive detection of the luminescence decay of 1O2, produced using two common photosensitizers, Rose Bengal and Hypericin, within the 60-m diameter core of a 15-cm length of HC-PCF. We demonstrate the feasibility of directly detecting sub-picomole quantities of 1O2 using this methodology, and identify some aspects of the HC-PCF technology that can be improved to yield even higher detection sensitivity

Bioluminescence-Activated Photodynamic Therapy for Luciferase Transfected, Grade 4 Astrocytoma cells in vitro

BACKGROUND: Grade 4 astrocytoma is incurable due to the diffusely infiltrative nature of the disease. Photodynamic therapy (PDT) is a promising therapeutic option, but external light delivery is not feasible when cancer cells infiltrate unknown areas of normal brain. Hence the search for endogenous sources such as bioluminescence that can generate light at cancer cells. This requires a substrate (a luciferin) and an enabling enzyme (a luciferase), neither seen in mammalian cells. METHODS: Preliminary studies confirmed that U87 cells (derived from a human grade 4 astrocytoma) could be killed by conventional PDT using the photosensitisers hypericin or mTHPC. U87 cells were then transfected with firefly and other luciferases and light generating cell lines (U87-luc, U87-hRluc, U87-CBG68luc) identified using the appropriate substrate. Reagent doses and conditions were optimized and U87-luc cells incubated with hypericin or mTHPC with d-luciferin added to initiate bioluminescence activated PDT (bPDT). Cell survival was assessed by MTT assay, haemocytometry and growth assay. Control groups included U87-luc cells with no added active reagents, substrate only, photosensitiser only and non-transfected U87 cells. Results were expressed as a percentage of surviving cells compared with untreated U87-luc controls. RESULTS: There was no bPDT effect on non-transfected cells. The mean survival of treated transfected cells was 36%, (P<0.001) using hypericin and 35% (P<0.001) using mTHPC, compared with untreated U87-luc cells. bPDT effects were suppressed by the anti-oxidant, lycopene. CONCLUSIONS: bPDT can kill Grade 4 astrocytoma cells transfected with luciferase in vitro. This justifies progression to in vivo studies

Cathepsin B-degradable, NIR-responsive nanoparticulate platform for target-specific cancer therapy

Stimuli-responsive anticancer formulations can promote drug release and activation within the target tumour, facilitate cellular uptake, as well as improve the therapeutic efficacy of drugs and reduce off-target effects. In the present work, indocyanine green (ICG)-containing polyglutamate (PGA) nanoparticles were developed and characterized. Digestion of nanoparticles with cathepsin B, a matrix metalloproteinase overexpressed in the microenvironment of advanced tumours, decreased particle size and increased ICG cellular uptake. Incorporation of ICG in PGA nanoparticles provided the NIR-absorbing agent with time-dependent altered optical properties in the presence of cathepsin B. Having minimal dark toxicity, the formulation exhibited significant cytotoxicity upon NIR exposure. Combined use of the formulation with saporin, a ribosome-inactivating protein, resulted in synergistically enhanced cytotoxicity attributed to the photo-induced release of saporin from endo/lysosomes. The results suggest that this therapeutic approach can offer significant therapeutic benefit in the treatment of superficial malignancies, such as head and neck tumours

Apoptotic cell death induced by dendritic derivatives of aminolevulinic acid in endothelial and foam cells co-cultures

Photodynamic therapy (PDT) is an effective procedure for the treatment of lesions diseases based on the selectivity of a photosensitising compound with the ability to accumulate in the target cell. Atherosclerotic plaque is a suitable target for PDT because of the preferential accumulation of photosensitisers in atherosclerotic plaques. Dendrimers are hyperbranched polymers conjugated to drugs. The dendrimers of ALA hold ester bonds that inside the cells are cleaved and release ALA, yielding PpIX production. The dendrimer 6m-ALA was chosen to perform this study since in previous studies it induced the highest porphyrin macrophage: endothelial cell ratio (Rodriguez et al. in Photochem Photobiol Sci 14:1617-1627, 2015). We transformed Raw 264.7 macrophages to foam cells by exposure to oxidised LDLs, and we employed a co-culture model of HMEC-1 endothelial cells and foam cells to study the affinity of ALA dendrimers for the foam cells. In this work it was proposed an in vitro model of atheromatous plaque, the aim was to study the selectivity of an ALA dendrimer for the foam cells as compared to the endothelial cells in a co-culture system and the type of cell death triggered by the photodynamic treatment. The ALA dendrimer 6m-ALA showed selectivity PDT response for foam cells against endothelial cells. A light dose of 1 J/cm2 eliminate foam cells, whereas less than 50% of HMEC-1 is killed, and apoptosis cell death is involved in this process, and no necrosis is present. We propose the use of ALA dendrimers as pro-photosensitisers to be employed in photoangioplasty to aid in the treatment of obstructive cardiovascular diseases, and these molecules can also be employed as a theranostic agent.Fil: Céspedes, Mariela Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Saenz, Daniel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Calvo, Gustavo Hernán. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Gonzalez, Marina Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: MacRobert, Alexander J.. University College London; Reino UnidoFil: Battah, Sinan. University Of Essex; Reino UnidoFil: Casas, Adriana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Di Venosa, Gabriela Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentin